JCS021394 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Depletion of the anti-K18 antibody. The anti-K18 antibody was pre-absorbed with 100 µg recombinant K18. Antibodies before and after absorption were used for immunostaining and examined by epifluorescence microscopy. Images were taken under the same exposure condition.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Calculation of the percentage of eIF3k associated with K18 in vivo. HeLa cells were lysed with Empigen lysis buffer and aliquots of lysate (S) and pellet (P) were used for western blot analysis with anti-K18 antibody (middle panel). A portion of lysate was used for immunoprecipitations with anti-K18 antibody or control antibody, and then analyzed by western blot with anti-K18 (middle panel) or anti-eIF3k (top panel) antibody as indicated. A portion of total cellular lysate was included for analyzing the total cellular amount of eIF3k (left lane, top panel). The relative intensity of each band corresponding to eIF3k or K18 was quantitated and indicated below. The amount of lysate or IP product loaded in each lane relative to the total amount used in this experiment is indicated as ‘% input’. The percentage of eIF3k associated with K18 was calculated using the algorithm described in the Materials and Methods. As indicated in the bottom panel, such experiment was repeated for three times and an average of 16.5±6.4% was obtained (13.1% for the experiment shown in this figure).

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. eIF3k does not affect the integrity and organization of K8/K18 filaments. (A) HeLa cells were transiently transfected with FLAG-eIF3k. (B) HeLa cells stably expressing eIF3k siRNA and GFP (see Fig. 5A for description) were mixed with parental HeLa cells. For cells in both A and B panels, they were immunostained with antibodies to eIF3k, K18 or Flag as indicated and then examined by confocal microscopy. One confocal section corresponding to each individual labeling is shown. Cells expressing eIF3k siRNA are marked by an arrow.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Downregulation of eIF3k does not affect global translation and cell proliferation. (A) HeLa cells expressing eIF3k siRNA or control siRNA as shown in Fig. 5 were analyzed by BrdU cell proliferation assay. Cells were seeded at densities as indicated and then labeled with BrdU for 2 and 20 hours. (B) Cells as in A were metabolically labeled for various time points as indicated. The incorporation of ³⁵S-methionine and cysteine into polypeptide chains was analyzed.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. The formation of cytoplasmic inclusions and localization of eIF3k in these inclusions are both dependent on caspase activity. HeLa cells pre-treated with or without zVAD-fmk were irradiated with UV at 0.02 J/cm² and then incubated for 4 hours. Cells were fixed, stained with anti-eIF3k, anti-K8/K18 and Hoechst 33258, and examined by epifluorescence microscopy.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. Downregulation of eIF3k increases the association of active caspase 3 with K18. SW13-K8/K18 cells described in Fig. 7E were irradiated with UV at 0.015 J/cm². Cells were lysed as in Fig. 9E and lysates were used for immunoprecipitation with anti-K18 antibody. The immunoprecipitates and cell lysates were resolved by SDS-PAGE and then analyzed by western blot with antibodies as indicated.

• Supplemental Figure S7 (Adobe PDF) -

  Fig. S7. The distribution of overexpressed eIF3k. (A) HeLa cells were transfected with FLAG-eIF3k, double stained with anti-Flag and anti-K18 antibodies and then examined by confocal microscopy. The overlay of green and red images is shown in the ‘Merge’ panel. The overlap between FLAG-eIF3k and K18 image was analyzed using an EGD colocalization add-on to ImageJ software. (B) Parental HeLa cells (left) or HeLa cells transfected with FLAG-eIF3k were extracted with lysis buffer containing 0.5% Triton X-100 and the detergent-soluble and -insoluble fractions were collected as in Fig. 1F and analyzed by western blot with antibodies as indicated. As the FLAG-eIF3k was expressed at a much higher level than endogenous protein, a reduced amount of samples were loaded for the anti-eIF3k and anti-FLAG blots shown in the right panel to ensure an accurate quantitation.

• Movie 1 -

  Movie 1. Movie shows the 360° rotation of a 3D reconstructed image of active caspase 3 and M30 (inclusion body) distributions in HeLa cells carrying control siRNA as described in Fig. 9A.

• Movie 2 -

  Movie 2. Movie shows the 360° rotation of a 3D reconstructed image of active caspase 3 and M30 (inclusion body) distributions in eIF3k-depleted HeLa cells as described in Fig. 9A.